Solution gradients or density gradients are utilized in biochemical research to separate macromolecules such as proteins, DNA and RNA, and larger aggregates such as viruses and cells. More recently, density gradient centrifugation has found application in the field of nanotechnology. Researchers at Northwestern University have used gradients to separate and purify different classes of carbon nanotubes.
Solution gradients usually utilize a solute of varying concentrations to aid in the separation of particles. Examples of appropriate solutes are: sucrose, glycerol, CsCl, Optiprep™, Percoll™, ficoll, metrizamide, Nycodenz™ and/or sodium acetate. Particles are separated during centrifugation either by their velocity of sedimentation, or by their density if there is an isopycnic point within the solution column in the tube. Faster, or denser particles, respectively, will appear lower in the tube.
After the sample has been subjected into the appropriate density gradient in the centrifuge, the particles are recovered from the gradient for analysis. Fractionation methods and apparatus used to recover the sample in the gradient involve the transfer of the entire gradient or certain layers or bands of the solution gradient to other vessels. It is often desired to extract only desired bands from the solution gradient for electron microscopy, liquid scintillation or gel electrophoresis.
One of the earliest and simplest methods of fractionation is to pierce the bottom of the centrifuge tube with a fine bevelled needle and collect the drops of the solution gradient as it flows through the needle into a second vessel. The flow of the solution into the opening of the needle becomes conical. In other words, the particles directly in front of the needle opening and within a zone best described as an inverted cone above the needle are drawn into the needle opening before particles outside the cone. The resulting fractionation of different layers of the solution gradient significantly degrades the resolution achieved in the gradient.
Bottom puncture with side hole needles have also been used for fractionation. Side hole needles have a hole on each side of the needle tip. Side hole needles are more effective than the bevelled needle, but side hole needles also draw the solution into the needle in a conical fashion preventing high resolution of the fractionation.
One of the most common methods for fractionating solution gradients introduces a dense solution at the bottom of the centrifuge tube, which floats the gradient up to an inverted collection funnel placed on the top of the gradient. Some loss of resolution results from the retardation of particles near the tube wall during this upward movement, and at any but the slowest flow rates, the shallow collection cone fails to prevent the shallow collection cone fails to prevent the same cone-shaped extraction of liquid directly below the cone's central orifice experienced by the bevelled needle described above. The result is mixing of different layers in the gradient and the resultant loss of resolution.
These problems were addressed in U.S. Pat. No. 4,003,834 to Coombs, issued Jan. 18, 1977, an apparatus is disclosed for the fractionation of a solution gradient by displacement with a piston, and in U.S. Pat. No. 5,645,715 to Coombs issued 1995 which discloses a piston collection tip with a unique trumpet shape collection face. The use of a piston to displace the gradient from the top down solves the problem of particles adhering to the wall during the upward movement of the entire gradient since the gradient remains stationary until it is displaced by the downward movement of the piston. The trumpet tip prevents the cone-shaped mixing by gradually compressing horizontal bands into thin vertical columns prior to collection. Tubing carrying both air and rinse is disposed within the piston to allow for cleaning of the collection tubing, further improving resolution by preventing cross contamination between fractions. Pumping air into the piston tip transfers any solution gradient left in the tubing to a second vessel.
U.S. Pat. No. 4,003,834 also provides a means for visualizing bands of particles large enough to scatter visible light. However, many particles of interest are too small to scatter visible light or are present at too low a concentration to be detected. Since the nucleic acids and proteins found in these particles absorb UV light in the 260-280 nm range, it is the current practice to detect bands of these particles by passing the gradient outflow through a UV flow cell as is frequently done in HPLC and FPLC. The UV gradient profile obtained by the flow cell can be used as a diagnostic tool in its own right; however, in this application, the profile is generated as the gradient is being removed from the centrifuge tube.
There are two potential problems with this type of UV-based fractionation. Firstly, it is difficult to accurately and reproducibly identify the beginning and end of UV absorbance peaks (bands) in the profile as it is being generated. Secondly, unless the user manually interrupts the flow at the start and end of each peak, the fraction collector typically used to separate the gradient outflow into discrete fractions is doing so at a constant time interval or rate of flow. Thus, there is no relationship between the peaks of absorbance and the fractions and this requires the user to scan a range of fractions to identify those containing the particles of interest. Some UV-based collection systems have “peak-picking” algorithms built into their software so that rapid changes in UV absorbance in the outflow trigger sample collection into a new vessel. While providing adequate separation of discrete peaks of particles, these devices have difficulty detecting and separating overlapping peaks or shoulders. Volume- or time-based fractionation of the UV-flow cell output is disrupted by peak-picking, so the overall sampling profile is then lost. Thus, one must choose between obtaining uniform size samples for analysis or isolating peaks, as they are mutually exclusive.
Certain inventions (i.e. U.S. Pat. Nos. 4,873,875; 6,479,239) have attempted to obtain a UV or fluorescent profile of the contents of a centrifuged gradient by vertically scanning the gradient with a beam of light from the outside of the tube. These have not seen widespread use because the only centrifuge tubes that can withstand the severe stress of ultracentrifugation (100,000-1,000,000×g) are made of a UV-absorbing plastic, effectively preventing the beam from penetrating the tube. Consequently, the only devices currently capable of producing a UV profile of a gradient are those which pass the gradient through a detached UV flow cell.
It would be desirable, thus, to develop a means of generating a gradient profile independent of fractionation that may be used as a guide to fractionation.